Vancomycin-resistant monoclonal antibody hybridoma cell strain and application thereof

A technology of hybridoma cell lines and monoclonal antibodies, applied in the direction of anti-bacterial immunoglobulin, biochemical equipment and methods, material inspection products, etc., to achieve the effect of high detection sensitivity

Active Publication Date: 2015-12-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, microbiological assays are easily affe...

Method used

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  • Vancomycin-resistant monoclonal antibody hybridoma cell strain and application thereof
  • Vancomycin-resistant monoclonal antibody hybridoma cell strain and application thereof
  • Vancomycin-resistant monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Preparation of hybridoma cell line 2A1

[0021] 1. Synthesis of complete antigen

[0022] Take 7.0 mg vancomycin hydrochloride, add 2.0 mg EDC and 1.0 mg NHS, dissolve with 400 μLDMF, 200 μL MES buffer, stir at room temperature, activate for 4 hours; take another 5 mg BSA and dissolve it in 2 mL, 0.05 M, pH 9.6 CB solution, The above-mentioned activated vancomycin solution was slowly added dropwise to the BSA solution, stirred at room temperature overnight, dialyzed at 4°C for three days, and stored at -20°C to obtain the immunogen. Dissolve 5mg of vancomycin hydrochloride in water, add freshly prepared 2.5mgNaIO 4 Aqueous solution, activated in ice bath for 30min, slowly add the activated vancomycin solution dropwise to 5mgOVA solution dissolved in 2mL, 0.05M, pH9.6 CB solution, react at 4°C for 2h, then add 0.5mgNaBH 4 , reduced at 4°C for 2 hours, dialyzed at 4°C for three days, and stored at -20°C to obtain the coating agent.

[0023] 2. Animal immu...

Embodiment 2

[0032] Example 2 Application of Anti-Vancomycin Monoclonal Antibody

[0033] The monoclonal antibody prepared by hybridoma cell line 2A1 through in vivo ascites was applied to the ELISA addition and recovery test of vancomycin, and the specific steps were as follows:

[0034] (1) Coat the 96-well ELISA plate with 0.5 μg / mL original coating diluted with carbonate buffer solution (CBS), 100 μL per well, after coating at 37°C for 2 hours, wash the plate three times with PBST washing solution, 250μL per well each time, 3min each time, pat dry;

[0035] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 hours, wash the plate three times with PBST washing solution, 250 μL per well for 3 minutes each time, and pat dry;

[0036] (3) Prepare 0, 0.2, 0.5, 1, 2, 5, 10, 20ng / mL vancomycin in phosphate buffered saline (PBS); 0, 0.5, 1, 2, 5, 10, 20, 50ng / mL of norvancomycin standard solution. Add the standard solution and the extract of the sample to be te...

Embodiment 3

[0045] Example 3 Identification of subtypes of monoclonal antibody of hybridoma cell line 2A1.

[0046] Use the mouse monoclonal antibody Ig class / subclass identification enzyme-labeled secondary antibody kit to determine the subtype of the monoclonal antibody.

[0047] Table 12A1 Subtype Identification of Monoclonal Antibodies

[0048]

[0049] According to the test result of the kit, the subtype of the antibody is IgG2b.

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Abstract

The invention relates to a vancomycin-resistant monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection. The vancomycin-resistant monoclonal antibody hybridoma cell strain 2A1 is preserved in the General Microbiological Center of China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.10869; the vancomycin-resistant monoclonal antibody is produced by the hybridoma cell strain 2A1 with the preservation number of CGMCC No.10869 through secretion. The monoclonal antibody produced by the hybridoma cell strain through secretion has an IC50 (half maximal inhibitory concentration) value of 1.45 ng/mL to vancomycin, has an IC50 value of 3.98 ng/mL to demethylvancomycin, and further can be used for simultaneous detection of vancomycin and demethylvancomycin in food.

Description

technical field [0001] An anti-vancomycin monoclonal antibody hybridoma cell line and application thereof relate to the hybridoma cell line 2A1 and the anti-vancomycin monoclonal antibody secreted and produced, and belong to the field of food safety immunoassay. Background technique [0002] Vancomycin (VAN), whose English name is Vancomycin, is a glycopeptide antibiotic isolated from the fermentation broth of a strain of Amycolatopsis orientalis by McCormick et al. in 1956. It was approved by the United States in November 1958. Approved by the FDA, it was developed and marketed by Eli Lilly Company under the product name "Vancocin". The molecular formula of vancomycin hydrochloride is C 66 h 76 Cl 3 N 9 o 24 , CAS No. 1404-90-6, molecular weight 1485.71. In China, demethylvancomycin (NVAN) is mainly produced by actinomycete Dongfang No. 23, and its English name is Norvancomycin, which was put into clinical use in 1967. Both are used to treat infectious diseases cause...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/68G01N33/577
Inventor 匡华宋月鸿胥传来徐丽广马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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